Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We developed a protocol for quantification of relative gene expression using reverse transcription-polymerase chain reaction (RT-PCR) without the use of radioisotopes, special equipment or extra nucleotide fragments, such as competitors. The relative gene expression of GABA(A) receptor beta(1) subunit (GABA(A)Rbeta(1)) and phospholipase C beta(4) subtype (PLCbeta(4)) in rat cerebrum and cerebellum were determined by comparing the ratio of PCR products generated by linear amplification of the target cDNA segments and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA segment as a reference. The density of PCR products was measured from digitized images of photographs of ethidium-bromide-stained agarose gels. The linear region of PCR amplification was within the linear range (from 0.3 to 12 ng DNA in a single band) of the detection system. The accuracy of the present method was <2-fold difference in gene expression in a single determination and a 1.5-fold difference was statistically significant after repeated measurements. The estimated relative expression of PLCbeta(4) was significantly higher in cerebellum than cerebrum, and that of GABA(A)Rbeta(1) was the same in these two regions. Using the present method, it is possible to quantify several different subunits and subtypes of known ion channel, neurotransmitter receptor and intracellular signaling enzyme gene families.
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PMID:Quantification of relative mRNA expression in the rat brain using simple RT-PCR and ethidium bromide staining. 1093 41

Serotonergic neurotransmission in prefrontal cortex (PFC) has long been known to play a key role in regulating emotion and cognition under normal and pathological conditions. However, the cellular mechanisms by which this regulation occurs are unclear. In this study, we examined the impact of serotonin on GABA(A) receptor channels in PFC pyramidal neurons using combined patch-clamp recording, biochemical, and molecular approaches. Application of serotonin produced a reduction of postsynaptic GABA(A) receptor currents. Although multiple 5-HT receptors were coexpressed in PFC pyramidal neurons, the serotonergic modulation of GABA-evoked currents was mimicked by the 5-HT(2)-class agonist (-)-2,5-dimethoxy-4-iodoamphetamine and blocked by 5-HT(2) antagonists risperidone and ketanserin, indicating the mediation by 5-HT(2) receptors. Inhibiting phospholipase C blocked the 5-HT(2) inhibition of GABA(A) currents, as did dialysis with protein kinase C (PKC) inhibitory peptide. Moreover, activation of 5-HT(2) receptors in PFC slices increased the in vitro kinase activity of PKC toward GABA(A) receptor gamma2 subunits. Disrupting the interaction of PKC with its anchoring protein RACK1 (receptor for activated C kinase) eliminated the 5-HT(2) modulation of GABA(A) currents, suggesting that RACK1-mediated targeting of PKC to the vicinity of GABA(A) receptors is required for the serotonergic signaling. Together, our results show that activation of 5-HT(2) receptors in PFC pyramidal neurons inhibits GABA(A) currents through phosphorylation of GABA(A) receptors by the activation of anchored PKC. The suppression of GABAergic signaling provides a novel mechanism for serotonergic modulation of PFC neuronal activity, which may underlie the actions of many antidepressant drugs.
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PMID:Serotonin receptors modulate GABA(A) receptor channels through activation of anchored protein kinase C in prefrontal cortical neurons. 1151 39

The protein p130 was isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-delta1 but lacking PLC activity. We show that p130 plays an important role in signaling by the type A receptor for gamma-aminobutyric acid (GABA). Yeast twohybrid screening identified GABARAP (GABA(A) receptor-associated protein), which is proposed to contribute to the sorting, targeting or clustering of GABA(A) receptors, as a protein that interacts with p130. Furthermore, p130 competitively inhibited the binding of the gamma2 subunit of the GABA(A) receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl- current by Zn2+ or diazepam, both of which act at GABA(A) receptors containing gamma subunits, is impaired in hippocampal neurons of p130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that p130 is essential for the function of GABA(A) receptors, especially in response to the agents acting on a gamma2 subunit.
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PMID:Role of the PLC-related, catalytically inactive protein p130 in GABA(A) receptor function. 1186 28

Application of group I metabotropic glutamate receptor (mGluR) agonists elicits seizure discharges in vivo and prolonged ictal-like activity in in vitro brain slices. In this study we examined 1) if group I mGluRs are activated by synaptically released glutamate during epileptiform discharges induced by convulsants in hippocampal slices and, if so, 2) whether the synaptically activated mGluRs contribute to the pattern of the epileptiform discharges. The GABA(A) receptor antagonist bicuculline (50 microM) was applied to induce short synchronized bursts of approximately 250 ms in mouse hippocampal slices. Addition of 4-aminopyridine (4-AP; 100 microM) prolonged these bursts to 0.7-2 s. The mGluR1 antagonist (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY 367385; 25-100 microM) and the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP; 10-50 microM), applied separately, significantly reduced the duration of the synchronized discharges. The effects of these antagonists were additive when applied together, suggesting that mGluR1 and mGluR5 exert independent actions on the epileptiform bursts. In phospholipase C beta1 (PLCbeta1) knockout mice, bicuculline and 4-AP elicited prolonged synchronized discharges of comparable duration as those observed in slices from wild-type littermates. Furthermore, mGluR1 and mGluR5 antagonists reduced the duration of the epileptiform discharges to the same extent as they did in the wild-type preparations. The results suggest that mGluR1 and mGluR5 are activated synaptically during prolonged epileptiform discharges induced by bicuculline and 4-AP. Synaptic activation of these receptors extended the duration of synchronized discharges. In addition, the data indicate that the synaptic effects of the group I mGluRs on the duration of epileptiform discharges were mediated by a PLCbeta1-independent mechanism.
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PMID:Role of synaptic metabotropic glutamate receptors in epileptiform discharges in hippocampal slices. 1236 93

GABA(A) receptors are critical in controlling neuronal activity. Here, we examined the role for phospholipase C-related inactive protein type 1 (PRIP-1), which binds and inactivates protein phosphatase 1alpha (PP1alpha) in facilitating GABA(A) receptor phospho-dependent regulation using PRIP-1-/- mice. In wild-type animals, robust phosphorylation and functional modulation of GABA(A) receptors containing beta3 subunits by cAMP-dependent protein kinase was evident, which was diminished in PRIP-1-/- mice. PRIP-1-/- mice exhibited enhanced PP1alpha activity compared with controls. Furthermore, PRIP-1 was able to interact directly with GABA(A) receptor beta subunits, and moreover, these proteins were found to be PP1alpha substrates. Finally, phosphorylation of PRIP-1 on threonine 94 facilitated the dissociation of PP1alpha-PRIP-1 complexes, providing a local mechanism for the activation of PP1alpha. Together, these results suggest an essential role for PRIP-1 in controlling GABA(A) receptor activity via regulating subunit phosphorylation and thereby the efficacy of neuronal inhibition mediated by these receptors.
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PMID:GABAA receptor phospho-dependent modulation is regulated by phospholipase C-related inactive protein type 1, a novel protein phosphatase 1 anchoring protein. 1530 41

In the present study, we investigated the effects of pentylenetetrazol (PTZ), a chemical convulsant that interacts with the GABA(A) receptor, in mice lacking the phospholipase C (PLC)-related inactive protein-1 (PRIP-1). PRIP-1 knockout mice did not develop spontaneous behavioral seizure. PRIP-1 knockout mice had markedly shorter latencies until the first clonic convulsion (CL) and tonic extensor (TE) following PTZ administration and increased incidence of convulsion compared to those in wild-type mice. Furthermore, the mortality rate by PTZ in mice lacking the PRIP-1 was also significantly increased in comparison with that in wild-type mice. These findings suggested that mice lacking the PRIP-1 were hypersensitive to PTZ-induced convulsion, and PRIP-1 might play roles in suppressing excessive excitability via interactions with the GABA(A) receptor.
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PMID:Hypersensitivity to pentylenetetrazol-induced convulsion in mice lacking the PLC-related inactive protein-1. 1546 66

Caloporoside is a natural active fungal metabolite, which was isolated from Caloporous dichrous and was described to exhibit antibacterial, antifungal and phospholipase C inhibitory activity. We have previously reported evidence that related beta-linked compounds, lactose and octyl-beta-d-mannoside, bind and functionally modulate rodent GABA(A) receptors, respectively. We have characterized the binding pharmacology of synthetic caloporoside and two further congeners, 2-hydroxy-6-([(16R)-(beta-d-mannopyranosyloxy)heptadecyl]) benzoic acid and octyl-beta-d-glucoside on GABA(A) receptors using a [35S]-t-butylbicyclophosphoorothionate (TBPS) radioligand binding assay. Caloporoside and 2-hydroxy-6-([(16R)-(beta-d-mannopyranosyloxy)heptadecyl]) benzoic acid produced concentration-dependent complete inhibition of specific [35S] TBPS binding with overall apparent IC50 values of 14.7+/-0.1 and 14.2+/-0.1 microM, respectively. In contrast, octyl-beta-d-glucoside elicited a concentration-dependent stimulation of specific [35S] TBPS binding (E(max)=144+/-4%; EC50=39.2+/-22.7 nM). The level of stimulation was similar to that elicited by diazepam (E(max)=147+/-6%; EC50=0.8+/-0.1 nM), and was occluded by GABA (0.3 microM). However, the three test compounds failed to elicit any significant effect (positive or negative) upon [3H] flunitrazepam or [3H] muscimol binding, indicating that they did not bind directly, or allosterically couple, to the benzodiazepine or agonist binding site of the GABA(A) receptor, respectively. The constituent monosaccharide, glucose, and both the closely related congeners octyl-beta-d-glucoside or hexyl-beta-d-glucoside have no significant effect upon [35S] TBPS binding. These data, together, provide strong evidence that a beta-glycosidic linkage and chain length are crucial for the positive modulation of [35S] TBPS binding to the GABA(A) receptor by this novel chemical class.
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PMID:Radioligand binding studies of caloporoside and novel congeners with contrasting effects upon [35S] TBPS binding to the mammalian GABA(A) receptor. 1616 65

Stroke neuroprotection trials suggest that pharmacological manipulations of a single neuroprotective mechanism are generally ineffective and that new approaches, possibly involving simultaneous manipulations of multiple mechanisms, need to be sought. To identify optimal components for such a multipronged approach, we studied NMDA receptor activation-induced cell death in organotypic hippocampal culture preparations as a model of excitotoxicity. Metabotropic group I glutamate receptor (mGluR) activation by their selective agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), resulted in concentration-dependent reduction of nerve cell susceptibility to NMDA-mediated injury (neuroprotective effect). The neuroprotection was mediated primarily by mGluR1, required phospholipase C activation, was inhibited by cholesterol-containing methyl-beta-cyclodextrin treatment, and occluded by antipsychotic quetiapine. It was associated with suppression of NMDA currents and prolongation of GABA(A) receptor-mediated currents in DHPG-treated cultures. cDNA microarray analysis of 1128 brain-relevant genes revealed that mGluR-mediated neuroprotection was associated with simultaneous activation of endocytosis, and inactivation of inflammation, cell adhesion, cell death, and transcription-related genes. Antisense inhibition of Rab5b, a gene coding for a small GTPase associated with endocytosis, significantly reduced the mGluR-mediated neuroprotection. These findings expand our understanding of the role that mGluRs play in regulation of nerve cell susceptibility to injury and should facilitate the design of novel therapeutic strategies for stroke and other neurodegenerative diseases.
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PMID:Activation of neuroprotective pathways by metabotropic group I glutamate receptors: a potential target for drug discovery? 1617 9

Dopamine modulates synaptic transmission in various brain regions. The disorder of dopamine system may be related to neurodevelopmental dysfunction. However, the action of dopamine on synaptic transmission during development is largely unknown. We studied the effect of dopamine on GABAergic and glutamatergic transmission in neonatal rat hippocampus from the early period of synapse formation by whole-cell patch-clamp recordings from CA1 pyramidal cells. Dopamine (100 muM) profoundly decreased the amplitude of GABA(A) receptor-mediated postsynaptic currents (GABA(A)-PSCs) to 32.2+/-5.4% (mean+/-S.E.M., EC(50): 2.9 muM) in the first postnatal week, when GABA provides excitatory drive. Dopamine also decreased the amplitude of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs) to 29.1+/-2.7% (EC(50): 18.7 muM) in the second postnatal week, when glutamate responses first appear. The dopamine-induced inhibition declined after these periods and became only partial after postnatal day 30. Further we identified the receptor subtype involved in the dopamine-induced inhibition as phosphatidylinositol-linked D1-like receptor, since 6-chloro-2,3,4,5-tetrahydro-3-methyl-1-(3-methylphenyl)-1H-3-benzazepine-7,8-diol hydrobromide (SKF 83959), a selective agonist for phosphatidylinositol-linked D1-like receptor, clearly mimicked the action of dopamine, and 1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), an inhibitor of phospholipase C, significantly reduced the dopamine-induced inhibition. Dopamine did not change the response to puff-applied GABA or kainic acid, nor the amplitude of miniature GABA(A)-PSCs or miniature EPSCs. These results suggest that the activation of phosphatidylinositol-linked D1-like receptor profoundly suppresses the excitatory transmission during the early period of synapse formation in the developing hippocampus by presynaptic mechanisms. This study firstly demonstrates the effect of phosphatidylinositol-linked D1-like receptor on synaptic transmission.
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PMID:Dopamine profoundly suppresses excitatory transmission in neonatal rat hippocampus via phosphatidylinositol-linked D1-like receptor. 1640 80

Cholecystokinin (CCK) interacts with two types of G protein-coupled receptors in the brain: CCK-A and CCK-B receptors. Both CCK and CCK-B receptors are widely distributed in the hippocampal formation, but the functions of CCK there have been poorly understood. In the present study, we initially examined the effects of CCK on GABA(A) receptor-mediated synaptic transmission in the hippocampal formation and then explored the underlying cellular mechanisms by focusing on the dentate gyrus region, where the highest levels of CCK-binding sites have been detected. Our results indicate that activation of CCK-B receptors initially and transiently increased spontaneous IPSC (sIPSC) frequency, followed by a persistent reduction. The effects of CCK were more evident in juvenile rats, suggesting that they are developmentally regulated. Cholecystokinin failed to modulate the miniature IPSCs recorded in the presence of TTX and the amplitude of the evoked IPSCs, but produced a transient increase followed by a reduction in action potential firing frequency recorded from GABAergic interneurons, suggesting that CCK acts by modulating the excitability of the interneurons to regulate GABA release. Cholecystokinin reduced the amplitude of the after-hyperpolarization of the action potentials, and application of paxilline or charybdotoxin considerably reduced CCK-mediated modulation of sIPSC frequency, suggesting that the effects of CCK are related to the inhibition of Ca(2+)-activated K(+) currents (I(K(Ca))). The effects of CCK were independent of the functions of phospholipase C, intracellular Ca(2+) release, protein kinase C or phospholipase A(2), suggesting a direct coupling between the G proteins of CCK-B receptors and I(K(Ca)). Our results provide a novel mechanism underlying CCK-mediated modulation of GABA release.
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PMID:Bidirectional modulation of GABAergic transmission by cholecystokinin in hippocampal dentate gyrus granule cells of juvenile rats. 1645 86


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